Nearly all multi-channel images show some degree of chromatic aberration. Chromatic aberration appears as focal shifts and possible scaling, rotational, and radial (2^nd, 4^th, 6^th degree) distortions between fluorescent channels. In addition, misalignment between light paths during acquisition can result in lateral (xy-direction) shifts between channels. These aberrations clearly affect any type of multi-channel analyses like colocalization and object-based analysis, and should therefore be fixed beforehand. The versatile Huygens Chromatic Aberration Corrector includes an easy-to-use Wizard that accurately estimates, visualizes and corrects up to 14 different chromatic aberration parameters.

Reliable analysis

Chromatic aberration significantly influences the results of multichannel analysis like colocalization, or distance measurements as part of object-based analysis. Huygens Chromatic Aberration Corrector offers an easy-to-use wizard to correct for serveral types of distortions.

Chromatic aberrations cause a broad set of distortions in addition to an xyz-shift, as exemplified in the TetraSpeck (Thermo Fisher Scientific Inc.) bead image below. A 3 parameter xyz-shift cross-correlation based correction is sufficient to effectively correct chromatic aberrations in the center of the image (compare panels g,h,i,j). However, higher order distortions can be observed when inspecting the image edges, as the green channel is shifted to the left in area 1, and to the right in area 3 (compare panels c and k). It is clear that a shift is not sufficient to correct for this. While additional correction of z-rotation and xy scale (5 parameters) already pose an improvement, the full set of 14 parameters is needed to fully account for the chromatic aberrations (compare panels d,e,f and l,m,n). Hence, while an xyz-shift is sufficient for a small field-of-view image, higher distortions must be accounted for in images with a larger field of view. Uniquely, Huygens offers the option to first determine the higher corrections from a bead image, save them to a template and then apply them to biological image data, even in batch mode.

Further note that with Huygens unique cross-correlation approach, an xyz-shift can often be estimated using the biological content of the image, averting the need for a separate bead calibration. When using this approach, it is advised to inspect the results carefully as a structural relationship between the content of each channel with the selected reference channel is assumed.

Broad set of distortions corrected

Huygens Chromatic Aberration Corrector Wizard helps you with selecting the right set of corrections to address misalignments (shifts), scaling and rotational differences, and radial (2^nd, 4^th, 6^th degree) distortions between channels. Radial deformations are caused by optical aberrations and appear as a pincushion or barrel distortion, or as a combination of both, also known as mustache distortion.

Use in research

Ivanova, D., Imig, C., Camacho, M. et al. CtBP1-Mediated Membrane Fission Contributes to Effective Recycling of Synaptic Vesicles.
Deconvolution and Chromatic Aberration Correction were performed with Huygens.
Cell Rep. 30, 2444–2459 (2020).

Dumbović, G., Sanjuan, X., Perucho, M. et al. Stimulated emission depletion (STED) super resolution imaging of RNA- and protein-containing domains in fixed cells.
Huygens Chromatic Aberration Corrector was used to estimate shift between channels.
Methods 187, 68-76 (2021).

For more, see Scientific Publications

Related products

Multi-channel images often suffer from crosstalk (a.k.a. bleed-through), which should be corrected before performing analyses. Huygens offers a wide range of analysis options, for example the assessment of the colocalization between different molecules.

Crosstalk Corrector Colocalization Analyzer

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More information

Point Spread Function
Huygens Deconvolution
Restoration examples
Chromatic Aberration